The present invention relates to a nucleic acid encoding a 1-2-rhamnosyl-transferase and uses thereof. More particularly, the present invention relates to a multistep process of converting hesperidin from orange peels to the sweetener neohesperidin dihydrochalcone (NHDC), in which, in one of the steps, 1-2-rhamnosyl-transferase in the presence of activated rhamnose is used in a rhamnosylation reaction to convert hesperidinase-treated hesperidin (H7G) to neohesperidin. Further particularly, the present invention relates to genetically modified plants of the Citrus genus including an antisense or sense (for co-suppression) construct which comprises the above nucleic acid or a gene knock-out integrated construct to provide less bitter grapefruits, pomelos and other citrus containing bitter flavanoid glycosides.
The bitter flavanones naringin and neohesperidin (FIG. 1) are produced only in young leaves and fruits of a few citrus species, such as grapefruit and pomelo, accumulate in a brief few week period, and remain through maturity (Castillo et al., 1992). Isomerically structured, yet tasteless flavanones, such as hesperidin (FIG. 1) are produced in oranges at the same time in development (Castillo et al., 1993). The differences between the tastelessness of orange hesperidin and the bitterness of grapefruit and pomelo naringin and neohesperidin depend on a specific set of glycosylation reactions (glucosylation and rhamnosylation) as further detailed hereinunder.
Low consumption of grapefruit is due in part to the bitter flavor of its juice and flesh (Matthews et al., 1990). This bitterness is due to the presence of large amounts of the flavanone glycoside naringin, as well as limonin in the juice. While limonin is a problem limited to the juice (Guadagni et al., 1973), in the intact fruit it appears in the tasteless A ring monolactone and forms the bitter dilactone only after maceration in the acidic juice environment (Matthews et al., 1990).
Thus, bitterness in the fruit can be decreased by reducing naringin levels. The bitterness in commercially prepared grapefruit juice is presently diminished to levels more acceptable by consumers by using resins that adsorb some bitter compounds or by treating the juice with the immobilized enzyme naringinase (Jimeno et al., 1987; Nikdel et al., 1989).
Commercial naringinase preparations typically consist of two enzymes, mainly xcex1-rhamnosidase and some xcex2-glucosidase, which successively hydrolyze one or both the sugar groups from naringin, leaving the tasteless compound prunin (naringenin-7-O-glucoside). Removal of the terminal rhamnose removes about 95% of the bitterness. Evidently, this procedure does not solve the problem of bitterness of whole fruit or home prepared juice.
A better approach which also addresses the above problem would, therefore, be to regulate the levels of naringin within the plant itself.
However, because of high heterozygousity of the commercial citrus varieties, classical plant breeding programs, will be hard put to yield identical varieties, yet having significantly less bitter fruit by reducing naringin. It will be appreciated in this respect that the presently available low naringin varieties (such as the Texas Red) are also low yielding.
While reducing the present invention to practice, studies of the naringin flavonoid metabolism (see below) assisted (i) in developing novel approaches to the problem; (ii) to further study the critical glycosylation steps that produce naringin; and (iii) in using this knowledge to modulate the degree of bitterness.
Citrus flavonoids ubiquity and biosynthesis: The genus Citrus contains many flavonoid glycosides that differ either in the structure of the aglycone or their sugar moieties. The major flavonoid in pomelo and grapefruit peel is naringin, while sweet orange peel contains hesperidin (Horowitz and Gentili, 1977 and FIG. 1). Peels of sour orange, trifoliate orange and Ponderosa lemon contain neohesperidin (FIG. 1). Neohesperidin and naringin are flavanone glycosides that contain the same disaccharide, xcex2-neohesperidose (2-O-xcex1-L-rhamnosyl-xcex2-D-glucose), which is attached at position C-7 of different aglycones, hesperetin and naringenin, respectively. Hesperidin and narirutin are isomers, respectively, of the above compounds that contain the disaccharide rutinose (6-O-xcex1-L-rhamnosyl-xcex2-D-glucose).
These flavonoids have some remarkable taste properties. Naringin and neohesperidin are extremely bitter, while narirutin and hesperidin are nearly tasteless (Horowitz and Gentili, 1986; Naim et al., 1986).
Flavonoids in Citrus: The highest naringin levels are associated with very young developing leaves and fruit tissue. Undifferentiated cells of Citrus paradisi (grapefruit) were able to biotransform exogenous naringenin and hesperetin, into prunin (naringenin-7-O-glucoside) and hesperetin-7-O-glucose (H-7-G), respectively. Further 1-2-rhamnosylation resulting in naringin or neohesperidin synthesis was not detected, although 1-6-rhamnosylation resulting in narirutin was observed (Lewinsohn et al., 1986, 1989b).
All of the above suggests that the blockage in the stepwise production of the naringenin from prunin (naringenin-7-O-glucoside) in the undifferentiated Citrus cells is caused by the absence of a specific rhamnosyl transferase activity.
Lewinsohn et al. detected chalcone-synthase and UDP-glucose, flavanone-7-O-glucosyl-transferase activities in cell-free extracts of Citrus (Lewinsohn et al. 1989a). Partial purification of the glucosyl transferase has been recently reported and some of its characteristics are therefore known (McIntosh and Mansell, 1989).
The glucosylated flavanone was further rhamnosylated. Chalcone-synthase activity was detected in cell-free extracts derived from young leaves and fruits.
Young fruits (2 millimeter diameter) had the highest chalcone synthase activity. In earlier studies it was shown that the glycosylation of the aglycone, naringenin, in undifferentiated Citrus cells occurs in two steps. First there was an initial glucosylation resulting in prunin (Lewinsohn et al. 1986, 1989b), and then a further rhamnosylation of prunin occurred when exogenous UDP-glucose and NADPH were added to the extract forming the end-product naringin (Lewinsohn et al 1989b).
Prunin was also shown to be a likely intermediate in the biosynthesis of naringin in immature grapefruit fruits (Berhow and Vandercook 1989).
Several glucosyl-transferases from plants catalyze the transfer of the sugar moiety from an activated UDP-sugar to a specific site on the flavonoids (Hahlbrock, 1981). Enzymatic preparations from plants catalyze the conversion of UDP-glucose to UDP-rhamnose in the presence of NADPH (Barber, 1962). The conversion is due to at least three different enzymatic activities requiring NADH or NADPH. It was not previously known how this conversion is catalyzed in Citrus. A coupled assay using UDP-glucose with NADPH was developed, which formed a rhamnosylated flavanone glycoside, indicative of a UDP-rhamnose:flavanone-7-O-glucoside-2xe2x80x2-O-rhamnosyl-transferase activity (xcex1-1-2 rhamnosyl transferase). A system was thereafter developed to directly biosynthesize radiolabeled UDP-rhamnose from [14C]-UDP glucose and NADPH for use in direct assay of the xcex1-1-2 rhamnosyl transferase activity during purification of the xcex1-1-2 rhamnosyl transferase. The direct assay facilitated the purification of the xcex1-1-2 rhamnosyl transferase. Once both rhamnosyl transferase substrates were available it was possible to purify the enzyme. Purity to homogeneity of the xcex1-1-2 rhamnosyl transferase in a four step procedure, which includes S-200 gel filtration, affinity column, ion exchange-FPLC and reverse-phase HPLC (Bar-Peled et al. 1991) was achieved and an antibody to it was elicited. These findings allowed to isolate the gene, which is the subject of this invention.
Semi-artificial sweetener: Neohesperidin and naringin from the peels of sour oranges and grapefruits are being converted into neohesperidin dihydrochalcone (NHDC), an intense sweetener used in the EU that is 2,000 fold sweeter than sugar (see right path on FIG. 1). NHDC is especially useful when long-lasting sweetness is desired, such as in chewing gum or in admixture with one of the artificial sweeteners. Currently, world-wide production of this NHDC is limited by the amount of neohesperidin and naringin available from these lesser grown Citrus species (less than 10% of total Citrus cultivated).
Unfortunately, the vast majority of citrus processed (oranges) contain large amounts of the hesperidin, an isomer having the terminal rhamnose attached by a 1xe2x86x926 linkage instead of the 1xe2x86x922 linkage. Hesperidin cannot be converted to the sweetener and is currently a waste by-product.
There is thus a widely recognized need for, and it would be highly advantageous to have, a nucleic acid encoding a 1-2-rhamnosyl-transferase to effect a multistep process of converting hesperidin from orange peels to the sweetener neohesperidin dihydrochalcone (NHDC), and to provide genetically modified plants of the Citrus genus including an antisense or sense (for co-suppression) construct thereof, or knock-out integrated construct, to provide less bitter grapefruits, pomelos and other citrus containing bitter flavanoid glycosides.
While reducing the present invention to practice, a novel rhamnosyl transferase gene from citrus responsible for producing the bitter flavanoids naringin and neohesperidin was isolated, sequenced, and characterized. This gene can be used in antisense or overproducing sense constructs to decrease the bitterness in grapefruit, and in sense constructs as part of the bioconversion of hesperidin from orange waste products to neohesperidin, used for the production of neohesperidin dihydrochalcone (NHDC), a sweetener. A knock-out construct can be used to knock-out the endo rhamnosyl transferase gene.
Thus, according to one aspect of the present invention there is provided an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having a flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase catalytic activity.
According to another aspect of the present invention there is provided a cell genetically modified to include, in an expressible sense orientation, a nucleotide sequence encoding a flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase.
According to still another aspect of the present invention there is provided a plant cell of a plant species naturally expressing a flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase messenger RNA, wherein the plant cell is genetically modified to include, in an expressible antisense orientation, a nucleotide sequence encoding an antisense RNA molecule being capable of in vivo base pairing with the flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase messenger RNA, to thereby render the flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase messenger RNA, when expressed, amenable to degradation by nucleases present in the plant cell.
According to yet another aspect of the present invention there is provided a transgenic plant of a species naturally expressing a flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase messenger RNA, wherein the transgenic plant is genetically modified to include, in an expressible antisense orientation, a nucleotide sequence encoding an antisense RNA molecule being capable of in vivo base pairing with the flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase messenger RNA, to thereby render the flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase messenger RNA, when expressed, amenable to degradation by nucleases present in the transgenic plant.
According to a further aspect of the present invention there is provided a plant cell of a plant species naturally expressing a flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase, wherein the plant cell is genetically modified to include, in an overexpressible sense orientation, a nucleotide sequence encoding a sense RNA molecule being capable of inducing a co-suppression effect to thereby reduce production of the flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase in the plant cell.
According to yet a further aspect of the present invention there is provided a transgenic plant of a species naturally expressing a flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase, the plant is genetically modified to include, in an overexpressible sense orientation, a nucleotide sequence encoding a sense RNA molecule being capable of inducing a co-suppression effect to thereby reduce production of the flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase in the plant cell.
According to still a further aspect of the present invention there is provided a transgenic plant or cell of a plant species naturally expressing a flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase, the transgenic plant or cell being genetically modified to knock-out a gene encoding said flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase to thereby abolish production of said flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase in the transgenic plant or cell.
According to an additional aspect of the present invention there is provided a commercial process of producing neohesperidin comprising the steps of: (a) extracting hesperidin from a hesperidin producing species of the genus Citrus; (b) treating the hesperidin with a hesperidinase, thereby obtaining hesperetin-7-glucoside; and (c) treating the hesperetin-7-glucoside with a flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase in the presence of activated rhamnose, thereby obtaining neohesperidin.
According to yet another additional aspect of the present invention there is provided a method of modifying a rhamnose-1-6-glucose linkage of a chemical compound to a rhamnose-1-2-glucose linkage comprising the steps of: (a) enzymatically treating the chemical compound to remove a rhamnose group thereof, thereby obtaining a first derivative of said chemical compound having a terminal glucose group; and (b) enzymatically treating the first derivative with recombinant flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase in the presence of activated rhamnose, thereby obtaining a second derivative chemical compound having the rhamnose-1-2-glucose linkage.
According to one further aspect of the present invention there is provided a commercial process of producing neohesperidin dihydrochalcone comprising the steps of: (a) extracting hesperidin from a hesperidin producing species of the genus Citrus; (b) treating the hesperidin with a hesperidinase, thereby obtaining hesperetin-7-glucoside; (c) treating the hesperetin-7-glucoside with a flavanone-7-O-glucoside-2xe2x80x3-O-rhamnosyl-transferase in the presence of activated rhamnose, thereby obtaining neohesperidin; (d) treating the neohesperidin with an alkali, thereby obtaining neohesperidin chalcone; and (e) reducing the neohesperidin chalcone, thereby obtaining neohesperidin dihydrochalcone.
The present invention successfully addresses the shortcomings of the presently known configurations by providing a nucleic acid encoding a 1-2-rhamnosyl-transferase to effect a multistep process of converting hesperidin from orange peels to the sweetener neohesperidin dihydrochalcone (NHDC), and to provide genetically modified plants of the Citrus genus to provide less bitter grapefruits, pomelos and other citrus containing bitter flavanoid glycosides.